Calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE) prepared from ovine and bovine brain display different maximal activities, amino acid compositions, and proteolytic fragments. An improved affinity chromatography purification procedure using pyridyldithioproprionyl CaM linked to thiol-Sepharose results in reproducible, quantitative elution of PDE activity. Dansyl-CaM, an active fluorescent derivative, exhibits two classes of CA++-binding sites; occupation of two higher affinity sites (Kd about 1 muM) corresponds to an increase in fluorescene and appears to involve cooperative CA-++ binding, whereas CA-++ binding to two lower affinity sites (Kd about 20-50 muM) parallels a decrease in fluorescence. The apparent affinity for CA-++ was increased at low Nacl concentrations while the decrease in flourescence intensity was not seen at elevated MgCl2 concentrations, suggesting Mg-++ competition for CA-++ sites. Interacion of dansyl-CaM with several binding proteins required occupation of the two high affinity CA-++ sites; their affinity was not altered in the presence of binding protein. These data suggest three distinct solution conformers of CaM, one of which [CaM.2(CA-++)] is sufficient for complex formation with PDE and several other CaM-binding proteins.